The present invention relates to a process for tumor treatment with cytokines, the monitoring of multidrug resistance (MDR)-associated genes and subsequent treatment with cytotoxic substances, as well as the use of these substances for producing a drug.
Efficiency of chemotherapy of malignant diseases is often limited by the simultaneous resistance towards structurally and functionally unrelated cytotoxic compounds. This multidrug resistance (MDR) phenomenon is mediated by MDR-associated genes such as mdr1, MRP (MDR-associated protein) and/or LRP (lung resistance protein). The classical resistance type of MDR is caused by overexpression of the mdr1 gene product, the P-glycoprotein (Pgp) (U. A. German et al., Sem. Cell. Biol. 4, (1993):63-76). In normal tissues Pgp expression has been detected mainly in epithelial cells with excretory or secretory function (A. T. Fojo et al., Proc. Natl. Acad. Sci. USA,84 (1987):265-269). In tumors of almost every localization, including in brain tumors, expression of the mdr1 gene has been observed (K. Nooter et al., Br. J. Cancer 63 (1991):663-669 and M. Mousseau et al., Eur. J. Cancer (1993):753-759). There exist a number of different approaches for the so-called reversal or overcoming of MDR that are mainly based on the inhibition of Pgp as drug-efflux-pump, but that also imply toxic side effects (J. Kellen, Anticancer Res. 13 (1993):959-961). Calcium channel blockers, alkaloids, Pgp-specific antibodies etc. are among these resistance modifying agents.
W. Walther and U. Stein describe in J. Cancer Research and Clinical Oncology (W. Walther et al., J: Cancer Res. Clin. Oncol. 120 (1994):471-478) and in British Journal of Cancer (U. Stein et al., Br. J. Cancer.47 (1996):1384-1391) the influence of cytokines on mdr1 expression in human colon carcinoma cell lines. It has been found, that 48 hours and 72 hours detectable reduction in mdr1 expression can be seen after cytokine treatment. The cell lines LoVo, SW480, LS174T, HCT15 and HCT116 were used for these experiments. TNFxcex1, IFNy and IL-2 were utilized as cytokines. Vincristine and doxorubicin were used as cytostatic substances. The cytokine-mediated effects on mdr1 gene expression, associated with chemosensitization, have also been shown for the gene transfer of TNFxcex1 into human U373 MG glioblastoma cells (W. Walther et al. Int. J. Cancer (1995):832-hours and 72 hours after cytokine treatment detectable reduction in mdr1 expression can be seen. The cell lines LoVo, SW480, LS174T, HCT15 and HCT116 were used for these experiments. TNFxcex1, IFNy, and IL-2 were utilized as cytokines. Vincristine and doxorubicin were used as cytostatic substances. The cytokine-mediated effects on mdr1 gene expression, associated with chemosensitization, have also been shown for the gene transfer of TNFxcex1 into human U373 MG glioblastoma cells (W. Walther et al. Int. J. Cancer (1995):832-8 839). It has been pointed out in agreement with other authors (e.g. C. H. Evans et al. Cancer Res. 52 (1992):5893-5899 and K. E. Sampson et al. Cancer Lett. 68 (1993):7-14), that modulation of mdr1/Pgp expression is an interesting alternative to conventional therapies for MDR reversal. A suitable procedure for this is neither proposed nor suggested.
It is an object of the present invention to provide a process for modulation of mdr1/Pgp expression. Using this process tumors can be treated, which until now were not or only minimally susceptible such a therapy with certain MDR-associated cytotoxic substances in chemotherapy. Thus, colorectal carcinomas would, in contrast to previous therapies with antimetabolites, be better susceptible therapies with MDR-associated cytostatics such as vinca alkaloids and/or anthracyclines.
The invention utilizes a cytotoxic, MDR-associated substance for the generation of a therapeutic for tumor therapy by chemosensitization of tumor cells of a cancer patient with a cytokine, the determination of the level of one or several MDR-associated genes (e.g. mdr1, Mrp, Lrp) in the tumor tissue of the patient in dependence on the start of the cytokine treatment, the determination of the time period during which the expression of one or several MDR-associated genes is considerably reduced by the cytokine treatment, the treatment of the patient during the determined time period (xe2x80x9ctherapeutic windowxe2x80x9d) with a therapeutically effective amount of a cytotoxic, MDR-associated substance, which is influenced (improved) in its efficacy by the expression of one or several MDR-associated genes.
Another object of the invention is a process for tumor therapy of patients with a MDR-associated cytotoxic substance, chemosensitization of tumor cells of a patient by cytokines, the determination of the level of one or more MDR-associated genes (e.g. mdr1, Mrp, Lrp) in the tumor tissue of the patient in dependence on the start of the cytokine treament, determination of the time period during which the expression of one or several MDR-associated genes is considerably reduced by the cytokine treatment, the treatment of the patient at the determined time period (xe2x80x9ctherapeutic windowxe2x80x9d) with a therapeutically effective amount of a cytotoxic, MDR-associated substance, which is influenced (improved) in its efficacy by the expression of one or several MDR-associated genes.
Thus, as used throughout the disclosure and the claims, xe2x80x9ctherapeutic windowxe2x80x9d means the patient-specific time period during which the expression of one or more MDR-associated genes is substantially reduced by the treatment with cytokine.
It was surprisingly found that after treatment of a cancer patient, a patient-characteristic time course of expression of one or several MDR-associated genes can be observed in his tumor cells. It appears that, after cytokine treatment a xe2x80x9ctherapeutic windowxe2x80x9d can be determined, which is patient-specific and which can be exploited to advantage for the subsequent cytostatic treatment. This is particularly of importance if the tumor tissue of the patient is removed and a cytostatic treatment will be performed for prevention of progression. It has been surprisingly shown, that also in these cases one can refer to that xe2x80x9ctherapeutic windowxe2x80x9d for the subsequent treatment with one or several MDR-associated cytotoxic substances, which has been determined before removal of the tumor.
It has been shown, that particularly gastrointestinal tumors, melanomas,, sarcomas, mammary carcinomas and glioblastomas can be treated with the procedure of the invention.
Under xe2x80x9ccytotoxic substancexe2x80x9d a substance is meant which is used as medicament for the treatment of malignant diseases and which acts on the basis of cytotoxicity mainly towards tumor cells. Such cytotoxic substances, which belong to the spectrum of multidrug resistance include for example daunorubicin, doxorubicin, mitoxantrone, et oposid, tenoposid, vinblastine, vincristine, actinomycin D, mitomycin C, taxol (paclitaxel), topotecan, colchicine, emetin, ethidiumbromide, puromycin, gramicidin D, valinomycin and mithramycin. Such substances and their mode of action are described e.g. by U. A. German et al. in European Journal of Cancer 32A (1996):927-944. Cis-platinum can likewise be suitable cytotoxic substances.
Under xe2x80x9ccytokinexe2x80x9d a substance is meant, which posses as cellular, physiological messenger immunstimulatory or also cytostatic properties and which can be used for cytotoxic or cytostatic tumor therapy. Suitable cytokines include for example interleukins such as IL-2, interferons, such as IFNxcex1 and IFNy and tumor necrosis factors such as TNFxcex1 etc.
Under xe2x80x9cmultidrug resistance-associated genexe2x80x9d (MDR-associated gene) a gene is meant, which codes for a protein causally involved in the development of the multidrug resistance phenotype, such as e.g. the energy-dependent transmembrane drug efflux pump (mdr1, U. A. German et al. Eur. J. Cancer 32A (1996):927-944; Mrp, D. W. Loe et al. Eur. J. Cancer 32A (1996):945-957) or which causes the nucleo-cytoplasmatic transport of a drug (e.g. Lrp, Izquierdo et al. Eur. J. Cancer 32A (1996): 979-984).
The expression of MDR-associated genes can be determined at both levels (mRNA and protein) with known molecular-biological techniques, e.g. with RT-PCR and/or Northern-hybridization using isolated RNA or mRNA, respectively, and/or with in situ RT-PCR in cryosections of the tumor samples. At the protein level the determination of MDR-associated gene products can be performed with e.g. Western-blotting using isolated proteins and/or with the method of immunohistochemistry in cryosections using the respective specific antibodies.
The time interval at which the expression of MDR-associated genes is considerably reduced can be determined by the time-dependent in vitro analysis of tumor samples, e.g. from biopsies taken daily (before treatment and 24, 48, and 72 hours after cytokine application); the above mentioned methods are employed..
Under xe2x80x9cconsiderablyxe2x80x9d or xe2x80x9csubstantially reducedxe2x80x9d is meant that if at least a two-fold reduction in mRNA and/or protein of the respective MDR-associated gene is determined.
Determination of expression of the MDR-associated genes is performed in the tumor tissue of the patient. For this, biopsies are taken. The mRNA of MDR-associated genes can be determined by in situ RT-PCR and the respective gene products can be determined by immunohistochemistry in cryosections. Beside these statements on heterogeneity of tumor cell areas and on expression of these genes in tumor cell areas, the level of expression of the genes is determined by quantitative methods (e.g. RT-PCR with isolated RNA) using external and internal standards.
The cytokine treament can be performed by using one cytokine, suitably IL-2, IFNxcex1, IFNy or TNFxcex1 or combinations of them, preferabely TNFxcex1/IFNy or IFNxcex1/IL2.
The treatment can be performed systemically or locally (e.g. perfusion of cytokines or cytokine-gene-therapy). It is preferred in the first step to treat the tumor tissue of the patient systemically or locally with one cytokine or combinations of them and in the last step to use the cytotoxic substance or their combinations locally or systemically. The cytokine is used at therapeutic concentrations, which will chemosensitize the tumor cells.
The cytokine is locally applied intratumorally, for which the cytokine itself or a nucleic acid, mediating the expression of the desired cytokine gene in the tumor cell, is utilized (e.g. local gene therapy, gene gun).
The cytotoxic substance is used at a therapeutically efficient concentration, which is destroying tumor cells in vivo to an appreciable extent.
The following example and the aforementioned publications explain the present invention. The process of the following examples is an exemplary illustration which can be modified.